This invention relates to methods and compositions for arousing dormant bacteria. More particularly, this invention relates to methods and compositions for arousing dormant bacteria for the purpose of detecting their presence, evaluating their threat to health, and, if warranted, killing them. Even more particularly, the invention relates to arousing dormant bacteria by altering their internal osmolality and/or pH and allowing adjustment time for the arousal mechanism to be initiated.
Dormant populations of bacteria are formed during the normal growth cycles of non-spore forming genera. Dormant bacteria were first described in 1925 as cells formed during the stationary phase of normal microbial growth (Burke, Sprague and Barnes, J. Inf. Dis., 36:555-560 (1925)). By definition, they do not grow on nutrient-rich media selected for their proliferation and classification. Since growth on selected media defines viability, confusion and disbelief continue to surround the existence of dormant bacteria. The phrase xe2x80x9cviable but not culturablexe2x80x9d (VBNC) is frequently used as a descriptor.
Dormancy is different from sporulation. Late in the stationary phase vegetative cells of the genera Bacillus and Clostridium form spores, dense particles resistant to adverse conditions. For example, Clostridial spores can withstand 100xc2x0 C. for more than one hour, therefore, saturated steam at 121xc2x0 C. for 15 minutes is required to kill them. Germination of these spores is induced by heating neutral suspensions of these cells at 65xc2x0 C. for 20 minutes. If L-alanine, L-tyrosine, or adenosine is added to the buffer, 100% of the spores will germinate.
Dormant bacteria are believed to form in response to environmental stressors that are not part of their normal growth cycle. Dormant bacteria form during the normal growth cycle for both harmless and pathogenic isolates of Gram-positive and Gram-negative bacteria Thus these non-detectable dormant bacteria have the potential to be widespread threats to human health. Their presence is suspicioned in recurring illness in patients and endemics. The spread of cholera has been in part attributed to dormant forms of V cholera in potable water. Brayton, P. R., et al. (1987) Appl. Envir. Micro. 53:2862. The outbreak of Legionella infections in Philadelphia in 1976 is believed to have resulted from the arousal of dormant L. pneumophila residing in a hotel""s air conditioning system. Steinert, M., et al. (1997) Appl. Envir. Micro. 63:2047. Endogenous dormant M. tuberculosis are thought to be responsible for recurrent tuberculosis. Hu, Y. M., et al. (1998) FEMS Micro LETT 158:139. Dormant Shigella dysenteriae produce diarrhea when orally administered to humans. Rahman, I., et al. (1994) Apl. Envir. Micro. 60:3573. Quiescent bacteria may be aroused during in vitro fertilization and embryo transfer. Peters, A. J., et al. Ob and Gyn 81:876 (1993).
Quiescent bacteria may be aroused during in vitro fertilization and embryo transfer. Peters, A. J., et al. Ob and Gyn 81:876 (1993).
Methods to xe2x80x9cresuscitatexe2x80x9d dormant bacteria have been identified for several genera or species, namely V. cholera, V. vulnificus, M. luteus, L. pneumophila, M. tuberculosis, and Nitrosomonas. Dormancy was induced by placing small amounts of vegetative cells into life-threatening conditions, such as nutrient or oxygen deprivation or low temperature environments for long periods, e.g. 125 days. Cells were resuscitated by restoring normal or rich conditions or by passing dormant bacteria through their parasitic or animal hosts. U.S. Pat. No. 5,314,542 discloses packaging bacteria from the genus Nitrosomonas in such a way as to induce dormancy, then reactivating these bacteria by increasing the concentration of their key nitrogen nutrient, ammonia, to 200 ppm and holding for 72 hours. To date, no one general method can be applied broadly to multiple species of bacteria.
For the foregoing reasons, a need exists for a generally-adaptable method to arouse dormant bacteria.
An object of this invention is to provide a method for arousing dormant bacteria.
Another object of this invention is to provide a method for recovering aroused, previously dormant bacteria from a culture for purposes such as classification, killing, or prevention of proliferation.
These and other objects, features, and advantages will become apparent after review of the following description and claims of the invention which follow.
This invention comprises a method for arousing dormant (non-spore forming) bacteria. The purposes of arousal include, for example, classification, killing, or prevention of proliferation in susceptible hosts.
Dormant bacteria are formed during the stationary phase of a normal growth cycle of both Gram-positive and Gram-negative species, both pathogenic and harmless species, and both feral and lab isolates. After proliferation, nonspore-forming bacteria may enter into a state of dormancy, and neither grow on media nor die in unfavorable environments. Although metabolically active, dormant bacteria are tolerant to antibiotics, chemicals, and other toxicants. Arousal can be induced by decreasing the internal osmolality, and thereby, the internal water activity of the cell (aw), or by neutralizing the internal pH.
The changes in osmolality or pH required for arousal are initiated by forcing the cell to diffuse its solutes or hydrogen ions into the media environment in a prescribed series of exposures to osmolal downshift gradients and providing for periods of adjustment. In other words, the internal osmolality of the dormant bacterial cell is decreased, and the cell is allowed to adjust to these changes gradually. During the adjustment period, the cell prepares to initiate replication. If the adjustment period is extended beyond that required for the initiation of growth, the cell can become hypermutative, as demonstrated by an ability to tolerate lethal doses of antibiotics without having been primed by exposures to less than lethal levels.
Different species require different adjustment periods and different rates of diffusion of internal solutes to maximize arousal. In addition, although just neutralizing the internal pH can be sufficient to induce arousal, an adjustment period of 10 days or more may be necessary to induce growth even in cultures that are easily aroused. When rates of diffusion and periods of adjustment are optimally controlled, the maximum number of dormant cells are aroused in the shortest amount of time. Too sudden diffusion from a gradient of too great an intensity will not induce arousal. On the other hand, if diffusion is too slow, arousal requires long periods of adjustment before arousal occurs.
The extent of downshift gradients and the length of the adjustment period required to arouse the maximum number of dormant cells in the shortest time is species dependent. Some, like L. monocytogenes is readily aroused, and others, like L. plantarum require extensive treatment before arousing. However, the present invention teaches a general method that arouses easily-arousable bacteria and provides guidance for arousing more difficult ones.
Definitions
xe2x80x9cDormantxe2x80x9d bacteria as used herein includes (a) bacterial cells that are xe2x80x9cviable but not culturablexe2x80x9d or xe2x80x9cquiescentxe2x80x9d or xe2x80x9cnascentxe2x80x9d which are (b) metabolically active, but (c) do not propagate in broths or on agar media formulated for their growth and identification.
xe2x80x9cArousingxe2x80x9d as used herein includes causing dormant cells to propagate in broths or an agar media developed for their growth and identification.
The xe2x80x9cvegetativexe2x80x9d form of dormant bacteria, as used herein, is that form of the dormant cells which grows on appropriate media.
xe2x80x9cHypermutativexe2x80x9d dormant cells are those that during arousal phase develop the ability to proliferate in the presence of lethal levels of an antibiotic to which they were not previously exposed.
Non-spore forming bacteria form dormant cells during the stationary phase of a normal growth cycle. Like spores, dormant cells proliferate in response to a specific physical change in their environment. Whereas heat provokes the biological change in spores that results in their germination, gradients of osmotic downshifts trigger the transformation of dormant cells to their vegetative forms and, if prolonged, to their hypermutative forms.
During the stationary phase, the cell accumulates small molecules, acids and other solutes, thereby increasing its osmotic pressure, lowering its water activity (aw) and, thus, halting cell division yet continuing some metabolic functions. The population of dormant cells in a duplex culture-a culture containing both living forms-can be as high as 107 colony-forming units (CFUs) per mL. In an older, xe2x80x9cnonculturablexe2x80x9d culture, dormant cells were found to average about 105 CFUs per mL. Arousal occurs when the high aw of the cell returns.
Subjecting bacteria or specimens to environments of osmolality of less than that of the cell followed by adjustment periods will arouse dormant bacteria. Such osmotic gradients can be accomplished any number of ways. Three preferred methods are
1. sequential diffusion (series of extractions),
2. progressive diffusion (sequential dilution), and
3. continuous diffusion (dialysis).
Although various buffers of osmolality less than that internal to the cell can be used, including media or those containing nutrients, the method has been standardized with phosphate buffered saline (Dulbecco""s PBS, pH 7.3, 0.8% NaCl)xe2x80x94a solution well known in microbiology.
This method was developed by first killing vegetative cells, inducing dormancy by extending the normal growth cycle of representative bacterial species, and then arousing feral and lab isolates of L. monocytogenes, L. caseii, L. plantarum, P. aeruginosa, and E. coli. By optimizing the parameters of downshift gradients of osmolality and the adjustment periods required for arousal, the general method developed can be adapted by one skilled in the art to assay any specimens taken from animals or terrestrial environments.
The arousal phase requires three periods: a downshift period, during which the internal osmolality is reduced at neutral pH, an adjustment period in which the cell prepares to initiate cell division, and a grow-out period on appropriate agar. The downshift gradients can be applied in a number of different ways depending on the skill of the operator and the convenience and availability of equipment. Some examples include:
1. a series of extractions over 20 minute time periods.
2. a series of sequential dilutions at increasing ratios from 1:5 to 1:1000.
3. dialysis against 250 volumes of buffer.
By applying these methods to test bacteria, the dormant forms are present in non-viable cultures at levels from 103 to 107 CFUs per mL. Viable cultures in their stationary phase can contain as many as 107 dormant cells, 0.1-10% of the viable population. Non-viable cultures may contain 105 dormant cells.
The preferred embodiment for progressive diffusion (dilution) is the 1:5 dilution, adaptive rest, 1:25 dilution, and 1:125 dilution. Four volumes of PBS, pH 7.3 are added to one volume of liquid specimen and allowed to adapt for a period of 24 hours at 37xc2x0 C. before being diluted twice again at 1:5. The 1:125 dilution is streaked on appropriate media to demonstrate viability and on agar plus antibiotic to demonstrate hypermutativeness.
The preferred method for sequential diffusion (extraction) is a series of 20 minute periods. The liquid specimen is microfuged. The pellet is suspended in PBS and allowed to diffuse for 20 minutes at 37xc2x0 C. before again microfuging and resuspending in fresh PBS. This is repeated up to 6 times and the suspension streaked on appropriate media with and without antibiotics.
The preferred method for continuous diffusion is as follows. For simple dialysis, the liquid specimen is placed in a washed dialysis bag with cutoff of 6-8 kDa and dialyzed against 250 volumes of PBS at 37xc2x0 C. with stirring for 40 minutes. The retentate is removed and allowed to adapt overnight before diluting 1:5 twice with PBS and streaking on appropriate agar. For centrifugal dialyis, the liquid suspension is centrifuged in a Centriplus(copyright) 10 tube (Millipore, Inc., Bedford, Mass.) at 3000 xg at room temperature for 30 minutes. The diffusate is removed and replaced with an equal volume of PBS added to the retentate and the centrifugation is repeated. The centrifugation and replacement of the diffusate is repeated
Aroused dormant cells become hypermutative if held for prolonged periods in the adjustment period during or after the downshift period. They can become tolerant to antibiotics and low-nutrient environments. Viable cells are not harmed by these forced diffusions, in fact, vegetative cells so treated can also become hypermutative.
In a mixture of vegetative and dormant cells, one can determine the levels and classification of dormant cells by first subjecting the mixture to killing conditions (e.g., heating at 100xc2x0 C.) and then arousing the dormant cells by following the above procedures of downshift gradients, adjustment, and grow-out periods.
After proliferation, non-spore forming bacteria can enter into a state of dormancy in which they are metabolically active but do not grow on appropriate media nor die when subjected to environments which kill their vegetative forms. A fundamental method to arouse dormant bacteria is useful for detection and classification and killing, if appropriate. On the other hand, useful food cultures can be kept xe2x80x9calivexe2x80x9d by avoiding conditions that arouse their dormancy.
Reduction of cells"" water activity (aw) and adjustment of the internal pH induces arousal. This is accomplished by
1. extracting solutes and H+ ions from the cell
2. allowing time to initiate the growth processes and the induction of mutativeness, and
3. providing time for their grow-out.
Arousal follows the suspension of dormant cells in an environment which induces the desorption of molecules from the cell. However, the rate of diffusion must be within a species-defined range, neither too severe or too gradual. For example, dormant cells of L. monocytogenes, a foodborne pathogen, are easily aroused by extracting the dormant cells in buffer for 20 minutes and growing them out overnight. They become hypermutative if extracted 3 times and allowed to adjust for 72 hours. Dormant cells of L. caseii, a dairy food culture, are aroused only if extracted 3 times, and do not become hypermutative. Dormant cells of L. plantarum, a harmless fresh vegetable organism, requires 7 extractions and an adjustment period of 144 hours to become aroused. Like L. caseii it appears not to possess the ability to become hypermutative.
Furthermore, dormant L. caseii is aroused if extracted with either 1xc3x97 or 4xc3x97 Dulbecco""s PBS, but not in water. The arousal mechanism is triggered by a lowering of the cell""s osmolality, but the optimum gradient is species-dependent.
In addition, dilutions of 1:5 may arouse more cells of a species than dilutions of 1:2 or 1:100 over the same time period. Some cells will be aroused after one dilution of 1:5 followed by an adjustment period of 24 hours. Other species may require additional dilutions, while still others require long adjustment periods, e.g., 72 hours.
Four subsequent dialyses against 250 volumes of buffer will arouse L. plantarum, but one dialysis against 1000 volumes will not.
Initiating hypermutativeness in all tested cells required adjustment periods longer than those required for arousal.
The length of time that a culture had been dormant did not appear to influence its conditions for arousal, nor did the temperature under which it became dormant, e.g., 4xc2x0 C., 25xc2x0 C., or 37xc2x0 C.
It is useful to employ the General Method in the first attempt to determine the presence of dormant bacteria. With results, refinements can be made as taught herein and claimed.
Arousal in the general method has three sequential periods:
1. a Downshift Period, in which the bacteria are placed in an environment that induces a gradual decrease in the cell""s internal osmolality at a constant rate. This is done by exposing the cells to solutions of lower osmolalities for specific periods of time by extraction, dilution, dialysis, or similar methods which expose the cells to the solution. Molecules diffusing out of the cell release the constraints on the arousal mechanism. Buffers of neutral pH are more effective than acidic ones.
2. an Adjustment Period, in which biological reactions are induced in the cell after reduced osmolality. Adjustment begins during the downshift period and continues through the subsequent grow-out period. Bacterial cells, both dormant and vegetative become hypermutative during an adjustment period of 72-140 hours.
3. the Grow-out Period is the elapsed time between plating and the appearance of colonies. Vegetative colonies can appear overnight; mutants require up to 160 hours at 37xc2x0 C. Gram-positive viable cells are not killed nor proliferate by being subjected to downshift gradients for periods of days. Like aroused cells, they become hypermutative during an extended adjustment period. However, Gram-negative cells appear to be sensitive to downshifting. Unless transferred to media within 36 hours after being extracted 7 times with PBS, viable cells of P. aeruginosa and E. coli die. However, dormant cells of these species are aroused after 7 extractions and an adjustment period of 72 hours. But now, being in the vegetative state, their survival time is also limited to 36 hours. One needs to, therefore, be mindful of these differences and how the general methods can optimize arousals on a species-specific basis. This differential can be used to determine the existence of dormant forms in the presence of vegetative forms of Gram-negative genera.
Since the arousal of dormant bacteria is species-specific and specimens of interest may contain unknown species and mixtures of dormant species, the general method should be used for initial analysis. Subjecting dormant cultures to sequential extractions has been selected as an efficient and effective arousal method for general use. The parameters chosen are those which were found to arouse all the test bacteria and provoke hypermutativeness in capable isolates. Subsequent analyses can then be performed by any of the examples or combinations of affecting downshift osmolar gradients.
1. centrifuge the bacterial pellet and resuspend in an equal volume of Dulbecco""s PBS, pH 7.3 for 20 minutes at 37xc2x0 C. Repeat for a total of 7 extractions. Hold aliquots of the 3rd, 6th, and 7th extractions at 37xc2x0 C. for adjustment.
2. after an adjustment period of 3 days and 6 days, streak the 3 extractions on appropriate agar media and incubate at 37xc2x0 C. Observe growth of aroused and hypermutative cells for 5 days.